Endotoxin is a lipopolysaccharide present in a cell wall of a Gram-negative bacterium and is the most typical pyrogen. If a transfusion, a medicine for injection, or blood contaminated with the endotoxin is introduced into the human body, the endotoxin may induce severe side effects such as fever and shock. Therefore, it has been required that the above-mentioned medicine or the like be kept so as not to be contaminated with endotoxin.
By the way, a limulus amoebocyte lysate (hereinafter, also referred to as “LAL”) contains serine proteases which are activated by endotoxin. In the case where LAL reacts with endotoxin, a coagulogen present in LAL is hydrolyzed into a coagulin by an enzyme cascade of the serine proteases activated depending on the amount of endotoxin, and the coagulin is associated with one another to form an insoluble gel. By using the characteristic of LAL, endotoxin can be detected with a high sensitivity.
Meanwhile, β-D-glucan is a polysaccharide which constitutes a cell membrane characteristic of a fungus. Measurement of β-D-glucan is effective, for example, for screening of infectious diseases due to a variety of fungi including not only fungi which are frequently observed in general clinical practices, such as Candida, Aspergillus, and Cryptococcus, but also rare fungi.
In measurement of β-D-glucan, by using the characteristic of a limulus amoebocyte lysate to coagulate (coagulate to form a gel) by β-D-glucan, β-D-glucan can be detected with a high sensitivity.
Various methods have been proposed as a method for detection or concentration measurement of a physiologically active substance derived from an organism (hereinafter, also referred to as a predetermined physiologically active substance) which can be detected by a limulus amoebocyte lysate, such as endotoxin or β-D-glucan. One of the methods is a semi-quantitative gelation method involving: leaving a mixture obtained by mixing a sample to be used for detection or concentration measurement of the predetermined physiologically active substance (hereinafter, also simply referred to as “measurement of the predetermined physiologically active substance”) with LAL to stand still; inverting the container after a lapse of a predetermined time period; and judging whether the sample has gotten gelation or not based on the presence or absence of dipping of the sample to examine whether or not the sample contains endotoxin at a certain concentration or higher. As other examples of the method, there are also given a turbidimetric method involving analyzing a sample by measuring the time course of the turbidity of the sample caused by gel formation by a reaction between LAL and the predetermined physiologically active substance, a colorimetric method using a synthetic substrate which is hydrolyzed by an enzyme cascade to develop a color, and the like.
In the case where the predetermined physiologically active substance is measured by the above-mentioned turbidimetric method, a mixture of a measurement sample and LAL is produced in a dry-heat-sterilized glass measurement cell. Then, gelation of the mixture is optically measured from the outside. However, the turbidimetric method may require a very long period of time of for gelation of LAL particularly in a sample containing the predetermined physiologically active substance at a low concentration. To solve the problem, a method which can measure the predetermined physiologically active substance in a short period of time has been required. As examples of the method, there has been proposed a laser light scattering particle counting method or a stirring turbidimetric method capable of forming fine gel-particles by stirring a mixture of a measurement sample and LAL using a magnetic stirring bar, for example, and determining the presence of the predetermined physiologically active substance in the sample in a short period of time based on the intensity of laser light scattered by the fine gel-particles or based on the intensity of light transmitted through the mixture.
The above-mentioned various methods have been developed to reduce a detection time period or measurement time period of the predetermined physiologically active substance or to improve measurement sensitivity. However, all the methods have both advantages and disadvantages, and it has been desired to further improve the methods in terms of reduction in measurement time period, increase in the sensitivity, and elimination of interfering substances.